Amalgamating the tools and techniques of molecular biology, genetics, biochemistry, and pharmacology, this competing renewal application offers an experimental paradigm to validate key components of the polyamine pathway of Leishmania and to implement a strategy directed toward drug discovery for the treatment of leishmaniasis. The proposal will continue to focus on arginase (ARG), ornithine decarboxylase (ODC), Sadenosylmethionine decarboxylase (ADOMETDC), and spermidine synthase (SPDSYN), the entire complement of polyamine biosynthetic enzymes in Leishmania. During the course of our investigations on polyamine metabolism in Leishmania we have: 1) isolated genes encoding all four of these proteins from either L. donovani or L. mexicana; 2) created and characterized ?ldodc, ?ldadometdc, and ?ldspdsyn knockouts in L. donovani and a ?lmarg knockout in L. mexicana; 3) produced large and replenishable quantities of LdSPDSYN and LmARG protein for biochemical analysis; and 4) generated monospecific antisera against LdODC, LdADOMETDC, and LdSPDSYN. The majority of our studies thus far have been performed with the insect vector or promastigote form of the parasite, and this application will now extend our characterization of the polyamine pathway to the amastigote or mammalian stage of the parasite, primarily in rodent models. This grant proposal follows up upon our pivotal finding that ?ldodc L. donovani are profoundly incapacitated in their ability to infect mice and initiates a process that will pharmacologically exploit the genetically validated LdODC enzyme. Specific Aim I has four components. First, we will determine whether putrescine, the product of LdODC, can reverse the compromised infectivity phenotype of ?ldodc parasites in mice. Second, we will determine whether the virulence defect of the ?ldodc null mutant extends to LdADOMETDC and LdSPDSYN deficiencies by assessing parasite burdens after infection of mice with the ?ldadometdc and ?ldspdsyn gene deletion mutants. Third, we will expand our infectivity studies on the ?ldodc knockout to Syrian golden hamsters, a rodent model of visceral leishmaniasis that more closely resembles the human disease. Finally, we will attempt to pharmacologically simulate the deleterious effects of a ?ldodc lesion on parasite infectivity in mice by administration of a-difluoromethylornithine (DFMO), an inhibitor of LdODC, in the drinking water. Specific Aim II entails the implementation of a cell-based screen of a large, structurally diverse chemical library in order to discover novel inhibitors of LdODC that could potentially serve as [unreadable]leads[unreadable] for drug development against Leishmania infections. The screen is based upon a yeast strain whose growth is contingent upon LdODC expression. The final Specific Aim of this submission is based on our observation that ?lmarg L. mexicana do not display the levels of reduced infectivity observed with the ?ldodc L. donovani. We propose, therefore, to ascertain whether the disparity in the abilities of ?lmarg L. mexicana and ?ldodc L. donovani to infect mice can be ascribed to species or gene differences.